Molluscicidal Activity of the Methanol Extract of Callistemon viminalis (Sol. ex Gaertner) G.Don ex Loudon Fruits, Bark and Leaves against Biomphalaria alexandrina Snails.

Iranian Journal of Pharmaceutical Research (2014), 13 (2): 505-514
Received: March 2012
Accepted: October 2012

Copyright © 2012 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services

Original Article

Molluscicidal Activity of the Methanol Extract of Callistemon viminalis
(Sol. ex Gaertner) G.Don ex Loudon Fruits, Bark and Leaves against

Biomphalaria alexandrina Snails

Ahmed A Gohar fl *, Galal T Maatooq", Sahar R Gadara", Walaa S Aboelmaaty" and Atef M El-Shazly*

^Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura
35516, Egypt. ^Parasitology Department, Faculty of Medicine, Mansoura University, Mansoura
35516, Egypt.

Abstract

Methanol extracts of Callistemon viminalis (Sol. Ex Gaertner) G.Don Ex Loudon fruits,
bark and leaves were tested for molluscicidal activity. Snails were collected and kept in
dechlorinated water under standard condition. Ten adults Biomphalaria alexandrina, of the
same size, were introduced in plastic acquaria for each experiment. The fruits, barks and leaves
were extracted with methanol and the methanol extracts were kept for testing as molluscicides.
Different extracts proved to have molluscicidal activity against the vector of schistosomiasis,
B. alexandrina snails. LC50 values for C. viminalis fruits, bark and leaves were 6.2, 32 and 40
ppm respectively. The C. viminalis fruits extract showed the highest effect against the tested
snails. Histopathological studies proved that the site of action of all tested extracts was localized
in the digestive system and hermaphrodite gland.

Keywords: Callistemon viminalis; Molluscicides; Biomphalaria alexandrina;
Histopathology; Snails.

Introduction

Currently, there is an increased attention
for the use of new molluscicides which are
highly effective, rapidly biodegradable, less
expensive, readily available and probably easily
applicable with simple techniques than synthetic
molluscicides. One of the new trends in the
biological control of vectors of diseases is testing
the toxicity of plant extracts, as alternatives to
chemical molluscicides, which proved to be
environmentally safe and have less residual
activity. There are many restrictions of using
toxic compounds (pesticides and molluscicides)
with fresh water. Therefore, the safety of plant

* Corresponding author:
E-mail: ahmedgohar99@yahoo.com

extracts to human being is an advantage for
studying their effect against the snail vectors of
schistosomiasis.

The botanical molluscicides are of economic
importance, especially in developing countries
(1). Also, there is a continuous need to search
for new plant species with ideal molluscicidal
properities (2-3). Different plants have been
reported as molluscicides (4-7). In Egypt,
screening of local plants for molluscicidal
activity has received increasing attention (8-13).

The treatment of B. alexandrina snails with
sublethal concentration of C. lanceolatus was
effective in altering the amino acid profile of this
snail species which could be contributed to the
impairment of snail's egg laying capacity, snail-
schistosome miracidiae finding mechanisms and
immune response of the molluscan hosts but has

GoharAe* al. I IJPR (2014), 13 (2): 505-514

no effect on the mammalian skin penetration
rate by schistosome cercariae (14). The genus
Callistemon was reported to contain diverse
chemical profile; steroids and triterpenes
(15 - 18), flavonoids (15, 19 and 20), tannins
and phenolic compounds (15, 19 and 21),
tetradecahydroxanthenediones (22), in addition
to the essential oils (23, 24). The diversity of
chemical constitution of different Callistemon
species reflects diversity in its biological
activities; antibacterial and antifungal activities
(25, 26), molluscicidal activity (14), Bio-
repellents for land leeches, insecticidal and
anthelmintic (27- 29), in addition to antioxidant
and hepatoprotective activity (21), antithrombin
(30), antidiabetic (31), anti-inflammatory (32),
anti- alzheimer's disease (20).

Reviewing the current literatures, C.
viminalis was not previously investigated for
mulloscicidal activity. So, the aim of the present
study is to evaluate the efficacy of the methanolic
extracts of C. viminalis (Sol. Ex Gaertner)
G.Don Ex Loudon fruits, bark and leaves as
molluscicides against Biomphalria alexandrina
snails. Some histological parameters in snail's
tissues were determined.

Experimental

Materials and methods
Collection of snails

Fresh water snails were collected by using
ordinary snail traps with long hand (33)
from the irrigation and drainage canals in
Mansoura, Dakahlia Governorate. The snails
were individually isolated and placed in plastic
bags with a suitable amount of water from the
same source and immediately transported to
the laboratory (34). The snails were identified
according to Ibrahim et al. (35).

Maintenance of snails

Laboratory bred B. alexandrina snails were
used in the work. The snails were washed several
times and maintained in dechlorinated tap water
to acclimatize to the laboratory conditions for
four weeks before experimentation (36). The
average size of the used adult snails was 10-14
mm in diameter. The snails were kept separately
in plastic aquaria, 8-10 liters capacity, in

dechlorinated tap water allowing 10 snails/
liter to avoid crowding. The aquaria contained
no mud, sand, gravel or any other substratum.
Dechlorinated water was obtained by storing
tap water in large containers for at least 48
hours up to one week. Water in the aquaria
was changed as frequent as required to keep
the snails in a good condition for experiments.
Dead snails were removed every day, and no
artificial aeration was used. The snails were
fed on fresh boiled lettuce leaves, and they
were kept under natural illumination. The
temperature was the ordinary room temperature
with its natural diurnal fluctuations (24-26 ±
2). Maintenance of snails was established in
Parasitology Department, Faculty of Medicine,
Mansoura University.

Plant materials

C. viminalis (Sol. Ex Gaertner) G.Don
Ex Loudon fruits, bark and leaves. The plant
identity was confirmed by Dr. Ibrahim Mashaly;
Professor of systematic botany, Faculty of
Science, Mansoura University, Egypt.

Preparation of plant extracts

The shade-dried fruits, bark and leaves (1
Kg, each) were powdered and extracted by
percolation in two liters percolator with distilled
methanol. The combined methanol extracts
of each part were concentrated to a syrupy
consistency under reduced pressure and then
allowed to dry in a desiccator over anhydrous
CaCl 2 . The dried extracts were kept for testing
as molluscicides.

Bioassay

Ten adults B. alexandrina of the same size
were introduced in plastic acquaria (25x13x7
cm) with the selected concentration of the
tested extract. Three grams of each extract
were transferred into 100 mL volumetric flasks,
and then diluted with dechlorinated tap water
to complete the volume. These solutions were
used to prepare the required concentrations.
Different concentrations of the tested extracts
(10,20 and 40 ppm) were used at three replicates
and three containers were left as control. The
results were recorded every 24 hours up to 5
days for the number of dead snails. Death of the

506

MoUuscicidal Activity of the Methanol Extract of Callistemon Viminalis

Table 1. Death of snails in the control experiment.

Time (day)

Number of snails -

Death

Control

%

1

30

1

3.3

2

30

2

6.7

3

30

2

6.7

4

30

3

10

5

30

3

10

snails was determined by lack of movement,
lack of response to gentle prodding with a blunt
object, discoloration of the shell, absence of
bleeding when they are crushed, and finally by
the standard method of immersing the snails
in a small amount of 5% sodium hydroxide
in a Petri-dish. If bubbles and blood come out
of the shell, it is recorded as alive, and Vice-
Versa (34, 37 - 39). After 24 hours, LC 50 values
for each extract were calculated according to
Litchfield and Wilcoxon (40). The percentage
of dead snails was calculated according to
Abbot>s formula, 1952:

, ., % killed by treatment x % dead in control

Killed snails =

1 00 - % dead in control

Histological preparations

Treated and control snails were removed
from their shells, washed thoroughly with
distilled water and fixed in 10% formalin.
The snail tissues were processed for paraffin
sectioning after embedded in paraplast at 50
°C. The 7 um sections were stained with iron
haematoxylin and eosin, and examined for
tissue changes with light microscopy.

Results and Discussion

Effect of the tested extracts on the snails
In Tables 1,2,3 and 4, the results of the effect
of different C. viminalis extracts are listed. LC 25
and LC 50 values for each extract were listed in
Table 5. Death of snails began after 24 hours
from application at low concentrations. In the
control experiment, one snail was died after 24
hours, while with the tested extracts at least 8
snails were died. This indicates the molluscicidal
activity of the titled plant. Complete death of
snails was observed after the third day with the
extracts of bark and leaves at concentrations
20 and 40 ppm, while a concentration 10 ppm
afforded 66.7% and 63.3% mortality for the
extracts of bark and leaves respectively. On
the other hand, complete death of snails was
observed after the first day with the fruits extract
even at a concentration 1 0 ppm. LC 50 values for
C. viminalis fruits bark and leaves were 6.2, 32
and 40 ppm respectively. Thus, C. viminalis
fruits extract has the highest potential to kill the
snails at low concentration and in short periods.
According to the World Health Organization of
plant molluscicide, screening must kill snails
after 24 hours in a concentration 1 00 ppm or less
at constant water temperature (39).

In the control test, 3 snails out of 30 were
dead during the period of the experiment 10%.

Table 2. Effect of C. viminalis fruits extract on B. alexandrina snails (number 30).

Time (day)

Number of

5 ppm

10 ppm

20 ppm

40 ppm

snails

Death

%

Death

%

Death

%

Death

%

1

30

10

33.3

30

100

30

100

30

100

2

30

17

56.7

30

100

30

100

30

100

3

30

21

70

30

100

30

100

30

100

4

30

26

86.7

30

100

30

100

30

100

5

30

29

96.7

30

100

30

100

30

100

507

GoharAe; al. I IJPR (2014), 13 (2): 505-514

Table 3. Effect of Callistemon viminalis bark extract on Biomphalaria alexandrina snails (number 30).

Number of

10 ppm

20 ppm

40 ppm

1 111 If ( (Id \ )

snails

Death

%

Death

%

Death

%

1

30

9

30

12

40

17

56.66

2

30

16

53.3

23

76.7

25

83.3

3

30

20

66.7

30

100

30

100

4

30

25

83.3

30

100

30

100

5

30

28

93.3

30

100

30

100

Table 4. Effect of C. viminalis leaves extract on B. alexandrina snails (number 30).

Time (day)

Number of

10 ppm

20 ppm

40 ppm

snails

Death

%

Death

%

Death

%

1

30

8

26.7

13

43.3

15

50

2

30

15

50

21

70

24

SO

3

30

19

63.3

30

100

30

100

4

30

23

76.7

30

100

30

100

5

30

27

90

30

100

30

100

These results are in agreement with the results
of Youssif et al. (41) who reported that the daily
mortality of B. alexandrina was 2.2%.

Histopathological changes

Treatment of snails with C. viminalis
fruits, leaves and bark extracts showed great
histopathological signs to the hermaphrodite
gland and the digestive tract of the snails. The
harmful histopathological changes were a
function of each extract concentrations. Treated

B. alexandrina with a concentration 40 ppm of

C. viminalis fruits extract showed large vacuoles
and degeneration in the hermaphrodite gland,
destruction in the follicular membrane and the
mature ovum showed losing of the nucleolus
(Figure 2).

Large vacuoles and great destruction was
observed in the digestive acini and the columnar
epithelial cells (Figure 13). In the digestive
epithelia, there observed large evacuated

Table 5. LC 50 values for C. viminalis extracts against B.
alexandrina snails.

C. viminalis extract

LC 50

Fruits

6.2

Bark

32

Leaves

40

epithelial cells (Figure 24). While treatment with
concentrations 20 and 10 ppm of C. viminalis
fruits extract showed small vacuoles and
little degeneration in the hermaphrodite gland
(Figure 3 and 4). The size of vacuoles was not
affected by the decreasing in concentrations of
the extract and the destruction in the digestive
acini and the columnar epithelial cells was less
affected (Figure 14 and 15). The evacuation
of the epithelial cells in the digestive epithelia
was moderate (Figure 25 and 26). On the other
hand, treatment with concentration 5 ppm of C.
viminalis fruits extract showed small vacuoles
in the hermaphrodite gland (Figure 5), normal
digestive acini and normal columnar epithelial
cells (Figure 16) and normal digestive epithelia
(Figure 27). This indicates that mild toxicity
afforded with the low concentration of the
fruit extract. Treated B. alexandrina with a
concentration 40 ppm of both C. viminalis bark
and leaves extracts showed small vacuoles and
moderate degeneration in the hermaphrodite
gland (Figures 6 and 9), small vacuoles in the
digestive acini and the columnar epithelial
cells (Figures 17 and 20) and small evacuated
epithelial cells in the digestive epithelia (Figures
28 and 31) while treatment with concentrations 20
and 10 ppm of both C. viminalis bark and leaves
extracts was comparable with that of 5 ppm of the

508

Molluscicidal Activity of the Methanol Extract of Callistemon Viminalis

Figure 1. T.S. in control B. alexandrina (Hermaphrodite
region). Anl= ancel's layer Sp= sperms S= spermatocytes 0=
oocyte X= 200.

fruits extract; small vacuoles in the hermaphrodite
gland (Figures 7, 8, 10 and 11), normal digestive
acini and normal columnar epithelial cells (Figures
18, 19, 21 and 22) and normal digestive epithelia
(Figures 29, 30, 32 and 33).

The tested plant extracts proved positive
molluscicidal activity against B. alexandrina
snails. It seems that the target tissues, for the

Figure 3. T.S. in treated B. alexandrina with 20 ppm fruits
extract (Hermaphrodite region). D: degeneration V: vacuoles

Figure 5. T.S. in treated B. alexandrina with 5 ppm fruits
extract (Hermaphrodite region). X=200.

Figure 2. T.S. in treated B. alexandrina with 40 ppm fruits
extract (Hermaphrodite region). D: degeneration V: vacuoles
X= 200.

tested extracts, were the hermaphrodite gland
and the digestive tract. Destruction of the
epithelial layer, vaculation and degeneration of
secretory cells are the histopathological signs
detected after treatment with the tested extracts.

References

Figure 4. T.S. in treated B. alexandrina with 10 ppm fruits
extract (Hermaphrodite region). D: degeneration V: vacuoles
X= 200.

Figure 6. T.S. in treated B. alexandrina with 40 ppm bark extract
(Hermaphrodite region). D: degeneration V: vacuoles X= 200.

509

GoharAe/ al. I IJPR (2014), 13 (2): 505-514

Figure 7. T.S. in treated B. alexandrina with 20 ppm bark
extract (Hermaphrodite region). V: vacules X= 200.

Figure 9. T.S. in treated B. alexandrina with 40 ppm leaves extract
(Hermaphrodite region). D: degeneration V: vacuoles X= 200.

Figure 13. T.S. in treated B. alexandrina with 40 ppm fruits
extract (digestive acini). X= 200.

Figure 8. T.S. in treated B. alexandrina with 10 ppm bark
extract (Hermaphrodite region). X= 200.

Figure 10. T.S. in treated B. alexandrina with 20 ppm leaf
extract (Hermaphrodite region). V: vacuoles X= 200.

Figure 12. T.S. in control B. alexandrina (digestive acini)
showing normal columnar epithelial cells. X= 200.

Figure 14. T.S. in treated B. alexandrina with 20 ppm fruits
extract (digestive acini). X= 200.

510

Molluscicidal Activity of the Methanol Extract of Callistemon Viminalis

Figure 15. T.S. in treated B. alexandrina with 10 ppm fruits
extract (digestive acini). X= 200.

Figure 17. T.S. in treated B. alexandrina with 40 ppm bark
extract (digestive acini). X= 200.

Figure 19. T.S. in treated B. alexandrina with 10 ppm bark
extract (digestive acini). X= 200.

Figure 21. T.S. in treated B. alexandrina with 20 ppm leaves
extract (digestive acini). X= 200.

Figure 16. T.S. in treated B. alexandrina with 5 ppm fruits
extract (digestive acini). X=200.

Figure 18. T.S. in treated B. alexandrina with 20 ppm bark
extract (digestive acini). X= 200.

Figure 22. T.S. in treated B. alexandrina with 10 ppm leaves
extract (digestive acini). X= 200.

511

GoharAe/ al. I IJPR (2014), 13 (2): 505-514

Figure 25. T.S. in treated B. alexandrina with 20 ppm fruits
extract showing digestive epithelia. A.E.c: evacuated epithelial
cells X = 200.

Figure 29. T.S. in treated B. alexandrina with 20 ppm bark
extract showing digestive epithelia. X = 200.

Figure 24. T.S. in treated B. alexandrina with 40 ppm fruits
extract showing digestive epithelia. A.E.c: evacuated epithelial
cells X = 200.

Figure 26. T.S. in treated B. alexandrina with 10 ppm fruits
extract showing digestive epithelia. A.E.c: evacuated epithelial
cells X = 200.

Figure 28. T.S. in treated B. alexandrina with 40 ppm bark
extract showing digestive epithelia. A.E.c: evacuated epithelial
cells X = 200.

Figure 30. T.S. in treated B. alexandrina with 10 ppm bark
extract showing digestive epithelia. X = 200

512

MoUuscicidal Activity of the Methanol Extract of Callistemon Viminalis

fe^ffi A.E.c

Figure 31. T.S. in treated S. alexandrina with 40 ppm leaves
extract showing digestive epithelia. A.E.c: evacuated epithelial
cells X = 200.

Figure 33. T.S. in treated B. alexandrina with 10 ppm leaves
extract showing digestive epithelia. X = 200.

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